Urothelium regeneration using viable cultured urothelial cell sheets grafted on demucosalized gastric flaps

OBJECTIVE: To evaluate urothelium regeneration by grafting viable cultured urothelial cell sheets, harvested from temperature-responsive culture surfaces, on demucosalized gastric flaps in a dog model. MATERIALS AND METHODS: Viable urothelium was obtained from eight beagle dogs by partial cystectomy. Harvested urothelial cells were seeded on temperature-responsive culture dishes modified with the thermally sensitive polymer, poly(N-isopropylacrylamide). Urothelial cells cultured for 3 weeks generated contiguous urothelial cell sheets that were noninvasively harvested with no enzymatic treatment from these dishes, by reducing culture temperature. Urothelial cell sheets were autografted onto surgically demucosalized gastric flaps. Three weeks after autografting the dogs were killed and the gastric flaps with the urothelial cell sheets were examined. Cell and tissue characteristics were compared between these urothelial cell sheet-grafted gastric flaps and native urothelium. Ultrafine structures were also examined by electron microscopy. RESULTS: Five of the eight urothelial cell sheet-grafted flaps showed viable urothelial regeneration. Urothelial cell sheets attached spontaneously to demucosalized tissue surfaces completely, with no suture or fixing, and developed into a stratified viable epithelium very similar to native urothelium. Regenerated urothelium remained unstained by antiproton pump antibody, which typically stains epithelial cells positively in gastric mucosal layers. On three of the eight flaps where there were severe haematomas, grafted cell sheets were not adherent and there was no urothelial regeneration. CONCLUSIONS: Urothelial cell sheets were autografted onto dog demucosalized gastric flaps successfully, with no suture or fixation, generating a multilayered urothelium in vivo. The novel intact cell-sheet grafting method rapidly produces native-like epithelium in vivo. This versatile technology should prove useful in urinary tract tissue engineering and surgical reconstruction.     

Isolation and primary culture of urothelial cells from normal human bladder

Summary Trypsinization (WT) was employed to disaggregate urothelial cells from normal human urinary bladder mucosa for the preparation of primary cultures. Urinary bladders of two male adults both 25 years old were obtained autopsy 1–2 h after death. The mucosa was incubated in HBSS containing 0.25% trypsin at 37°C. Mean cell yield, viability, and attachment were 14.6×10⁷, 76%, and 42.5% after WT. In histologic sections of treated mucosa, most of the urothelium was removed by WT. Following plating and attachment, cells obtained by WT formed a monolayer of flattened epithelial-like cells. When stained with polyclonal antikeratin antibody using the indirect immunoperoxidase technique, all of the cells were immunoreactive indicating an epithelial origin. In conclusion, based on morphology and immunocytochemistry, WT removed virtually all of the urothelial cells from the mucosa and no contamination by mesenchymal cells resulted from this procedure. Thus, WT is an appropriate technique for the isolation of urothelial cells and initiation of primary cultures of normal human urothelial cells for subsequent study.This work was supported by NIH Grant CA-28013     

Induction of the urothelial differentiation program in the absence of stromal cues

PURPOSE: The urothelial stroma is presumed to have a critical role in the formation and homeostasis of normal urothelium. To determine the intrinsic capacity of urothelial cells to initiate urothelial differentiation human urothelial cell were cultured under conditions that promote differentiation in the absence of stromal signaling. MATERIALS AND METHODS: Immortalized and primary human urothelial cells were cultured in semisolid medium. Recovered cells were then analyzed by immunofluorescence, flow cytometry and immunoblotting for expression of the differentiation specific keratins K18 and K8, and cyclin-cyclin-dependent kinase inhibitors. The expression of these markers in cells following semisolid culture was then compared with that in normal bladder and ureteral mucosa as well as in synthetic urothelium generated by 3-dimensional organotypic raft cultures. RESULTS: Organotypic raft culture of primary and immortalized urothelial cells generated full-thickness epithelium that resembled human bladder and ureteral urothelium, and expressed K8 and K18 in superficial layers. Suspension culture in semisolid medium induced K18 expression approximately 9-fold at 24 hours. p21 and p27 expression were induced by 6 hours and yet p21 expression subsided within 12 hours, while p27 expression persisted. CONCLUSIONS: These results indicate that primary and immortalized human urothelial cells have the capacity to enter the urothelial differentiation program and such entry does not require inductive signals from stroma. Furthermore, these data suggest that p21 and p27 have distinct roles in regulating the urothelial cell cycle.     

Investigations of urothelial cells seeded on commercially available small intestine submucosa

OBJECTIVES: To examine adherence and viability of human urothelial cells seeded on commercially available small intestine submucosa (SIS) specimens under serum-free conditions. MATERIALS AND METHODS: Before seeding, SIS was either washed with incubation medium or coated with collagen A, fibronectin, or pronectin. A possible influence of SIS itself on the viability of urothelial cells was analysed with conditioned cell culture medium obtained by incubation of SIS for 24hours. In addition, untreated SIS and a setting without SIS were used as controls. Viability of urothelial cells was analysed with the WST-1 assay until day 9. Histology of seeded and unseeded SIS specimens was investigated after Papanicolaou staining. To demonstrate urothelial cell adherence on SIS, immunohistology was performed with a mixture of monoclonal AE1 and AE3 anticytokeratin antibodies. RESULTS: Urothelial cells seeded on SIS revealed no measurable cell viability. SIS-conditioned cell culture medium was cytotoxic for urothelial cells after 24 hours. Histology only demonstrated cell nuclei and no cytoplasm both in seeded and unseeded SIS specimens, thus indicating porcine DNA. Expression of the cell type-specific marker proteins AE1/AE3 could not be demonstrated. CONCLUSION: Since the commercially available SIS specimens used contained porcine DNA residues and demonstrated cytotoxic effects on urothelial cells, SIS is not suitable for in vitro construction of urothelial cell-matrix implants.     

Hair follicle stem cells can be driven into a urothelial‐like phenotype: An experimental study

The aim of this study was to show that conditioned medium might induce transdifferentiation of hair follicle stem cells into urothelial‐like cells. Several conditioned media and culture conditions (skeletal muscle cell conditioned medium, smooth muscle cell conditioned medium, fibroblast conditioned medium, transforming growth factor‐conditioned medium, urothelial cell conditioned medium, and co‐culture of hair follicle stem cells and urothelial cells) were used. The hair follicle stem cells phenotype from rat whisker hair follicles was checked by using flow cytometry and immunofluorescence. Cytokeratins 7, 8, 15 and 18 were used as markers. Urothelial cell conditioned medium increased the expression of urothelial markers (cytokeratin 7, cytokeratin 8, cytokeratin 18), whereas it decreased a hair follicle stem cells marker (cytokeratin 15) after 2 weeks of culture. This process depended on the time of cultivation. This medium was able to sustain the epithelial phenotype of the culture. Other media including a co‐culture system failed to induce similar changes. Smooth muscle conditioned medium resulted in a loss of cells in culture. Hair follicle stem cells are capable of differentiating into urothelial‐like cells in vitro when exposed to a bladder‐specific microenvironment.     

Organ and species specificity in the stimulation of transitional epithelial cell growth by fibroblasts

OBJECTIVES: Culture of transitional epithelium for urinary tract reconstruction has been problematic due, in part, to the dependence of urothelial cells on a basal layer of bladder fibroblasts for growth. In vitro studies on the effect of bladder, ureter, and intestinal fibroblast cocultures and conditioned media upon urothelial cell growth were conducted to better characterize the dependence of urothelial cells of fibroblasts. METHODS: Primary cultures of human and porcine bladder, ureter, and intestinal fibroblasts and bladder and ureter urothelial cells were established. The urothelial cells were incubated with fibroblasts in a coculture system and growth compared to that of individual fibroblast and urothelial cultures. Urothelium-specific medium was exposed to the fibroblast cultures for 6, 12, and 24 h. Urothelial cell growth in each of the fibroblast-conditioned media was evaluated. RESULTS: Coculture of human urothelial cells with human bladder and ureter fibroblasts yielded increased cell growth when compared to the cells in individual culture. This improvement was greatest for the bladder fibroblasts in coculture with bladder epithelial cells. The media exposed to bladder and ureter fibroblasts for 24 h significantly increased bladder and ureter urothelial growth compared to fresh medium. Coculture with intestinal fibroblasts and exposure to intestinal fibroblast conditioned media did not significantly stimulate urothelial cell growth. Similarly, coculture and conditioned media studies of porcine urothelial cells with porcine bladder and ureter fibroblasts (but not intestinal fibroblasts) yielded increased cell growth when compared to the cells in individual culture, particularly with bladder fibroblasts. However, human urothelial cells were not stimulated by porcine bladder and ureter fibroblast conditioned media, nor were porcine urothelial cells stimulated by human bladder and ureter fibroblast conditioned media. CONCLUSIONS: The ability of urinary fibroblasts to stimulate urothelial cell proliferation resides in an unidentified soluble factor secreted into the medium, independent of the presence of the fibroblasts. This factor is relatively organ and species-specific.     

Immunoreactivity of p63 in monolayered and in vitro stratified human urothelial cell cultures compared with native urothelial tissue

OBJECTIVE: To investigate the immunoreactivity of p63 in monolayered and stratified human urothelial cell cultures and in normal urothelial tissues to assess the differentiation status of in vitro stratified urothelial constructs. METHODS: p63 expression was detected immunohistochemically in native normal human bladder, ureter, and renal pelvis tissues and immunocytochemically in monolayered urothelial cell cultures and urothelial constructs stratified in vitro. Additionally, expression of pancytokeratin, cytokeratin 20 (CK20), uroplakin III, and fibroblast surface antigen was investigated. RESULTS: In native tissues, urothelial cell layers showed the most intensive p63 staining in the basal cells; the superficial umbrella cells were predominantly negative. Monolayered urothelial cell cultures revealed reduced p63 expression with ongoing culture passages. In vitro stratified urothelial constructs exhibited p63 expression similar to that of native urothelium. CK20-reactive cells were absent in the monolayered cultures but present in the stratified cell cultures and in the urothelial constructs. In native urothelium, only superficial cells stained positive for CK20. Uroplakin III was not present in either monolayered urothelial cell cultures or stratified urothelial constructs. Cultured cells were always positive for pancytokeratin and negative for fibroblast surface antigen. CONCLUSIONS: p63 is a new biomarker for differentiation and stratification of urothelium created in vitro. For proposed clinical applications of in vitro stratified urothelium in reconstructive urology, urothelial constructs should exhibit expression of significant marker proteins similar to that of native urothelium. Our results show such similarity of expression for pancytokeratin, p63, and CK20, an encouraging possibility for confirming the functionality of tissue-engineered urothelia after clinical application.     

Bioartifizielles autologes Urothel etabliert aus Spülungen der Harnblase

Reconstructive surgery of lower urinary tract disorders can be limited by a shortage of adequate autologous tissue. Tissue engineering is an option for surgical reconstruction with evolved biological substitutes. Urethral repair with bioartificial urothelial implants can be an innovative method for sustained urothelial regeneration in situ. The needed urothelial cells are commonly isolated from native urothelium requiring surgery.The aim of this study was to establish primary human urothelial cell cultures from bladder washings in serum-free media and to generate urothelial tissue without seeding of matrices in a feeder cell-free system. It could be demonstrated that under these conditions bioartificial urothelium can be developed successfully from bladder washings. Its multilayered cellular structure and the initial differentiation in vitro, similar to native-grown urothelial tissue, are promising with regard to intended clinical application. Current work focuses on establishing cell culture techniques according to legal regulations, terminal differentiation of the urothelial constructs in vitro, and techniques to surgically implant lab-grown bioartificial urothelium.     

Epidermal growth factor receptor function in the human urothelium

Purpose

Epidermal growth factor receptor (EGFr)-targeted therapy may be used in subgroups of patients with urinary bladder cancer. Here we assessed the role of EGFr in urothelial proliferation and migration in a two- and three-dimensional cell culture system.

Methods

UROtsa cells derived from normal urothelium and malignant T24 cells were cultured in a Type I collagen gel. Proliferation and migration of urothelial cells, in the absence and presence of the EGFr inhibitor cetuximab, were assessed with a proliferation test (ATCC) and with the Axioplan 2 imaging microscope with a motorized stage (Carl Zeiss), respectively. The expressions of cytokeratin (CK) 17, CK20, EGFr, pEGFr, laminin, occludin and zonula occludens 1 (ZO-1) were assessed with immunohistochemistry and/or western blot.

Results

UROtsa spheroids were formed after 7 days in culture, while T24 cells did not form spheroids. UROtsa expressed CK20 but not laminin or CK17 and consequently resembled umbrella cells. In UROtsa and T24, cetuximab inhibited urothelial proliferation, induced cleavage of EGFr and/or pEGFR but did not affect urothelial migration. The tight junction protein occludin was cleaved, and the formation of cellular spheroids was inhibited in UROtsa by the presence of cetuximab.

Conclusions

EGFr modulates urothelial proliferation and the formation of the three-dimensional structure of the urothelium possibly by interfering with occludin. The present data also show a cell culture technique enabling phenotypically normal urothelial cells to form epithelial structures in contrast to malignant urothelial cells.

     

Serial cultivation of normal rat bladder epithelial cells in vitro

Recent advances in culture techniques for human urothelial cells have led to the development of an improved method for growing primary rat bladder epithelial cells. We report here the conditions developed for large-scale in vitro growth and serial cultivation of normal diploid rat bladder epithelial cells. Primary cultures were initiated by attachment of bladder mucosal explants to type I collagen gels. A rapid outgrowth of epithelial cells from the explants occurred when cultured in a hormone-supplemented medium with epidermal growth factor. These primary outgrowths were passaged by nonenzymatic dispersion with 0.1 per cent ethylenediaminetetracetic acid and replating onto new gels. The capacity for routine serial passaging and maintenance of rat bladder epithelial cells required the presence of epidermal growth factor, a requirement not observed with human urothelial cells. The characteristics of the cultured rat bladder epithelial cells were similar to human urothelial cells in: ultrastructural and phase-contrast morphologic properties, showing junctional complexes, desmosomes, stratification and an apical glycocalyx; the absence of stromal cell contamination; and the ability to be serially passaged. Spontaneous cell-line formation was observed with the rat bladder epithelial cells, but has not been found with the human urothelial cells. With the method that we have developed, the number of rat bladder epithelial cells generated from a single bladder of a 4 to 6 week old rat was increased 100-fold from about 7 X 10(5) cells to 7 X 10(7) viable cells within 3 weeks of culture. The capability of culturing normal, primary rat bladder epithelial cells on this scale has not been reported previously and will facilitate comparative studies of the biological and molecular characteristics of the mammalian urothelium. Furthermore, this culture system will be useful for carcinogenesis studies, including metabolic activation of carcinogens and cellular transformation in vitro.     

Bladder reconstruction using a prevascularized capsular tissue seeded with urothelial cells

PURPOSE: Recent advances in cell biology and tissue engineering have involved various avascular or acellular scaffolds with or without seeded cells. These techniques are frequently complicated by tissue necrosis, contracture and resorption. We used a vascularized matrix prelaminated with autologous cultured urothelial cells to reconstruct bladder wall defects. MATERIALS AND METHODS: A silicone block inserted into the right groin of 50 male Wistar rats directly superficial to the inferior epigastric vessels was used to induce capsule pouch formation. Urothelial cells harvested simultaneously and cultured were then suspended in fibrin glue and seeded into the newly formed capsule after removing the silicone block. After 1 week the prelaminated flap was transposed into a surgically created bladder wall defect. Experimental groups included rats with a urothelial cell seeded capsule pouch sacrificed at 1 and 4 weeks, respectively, after bladder reconstruction. In control rats scaffolds were treated only with fibrin glue or saline before transposition. RESULTS: Hematoxylin and eosin and immunohistochemical staining showed a continuous multilayered urothelial lining along the transposed prelaminated capsule flap in the experimental groups with better survival compared to controls treated only with fibrin glue (80% mortality) or saline (100% mortality). The surviving 3 control animals did not have a urothelial lining. CONCLUSIONS: Vascularized prefabricated flaps lined with culture derived urothelial cells were successfully used for bladder reconstruction in a rat model. The technique of prefabricating a vascularized scaffold lined with autologous urothelial cells may provide a method for future reconstruction of the genitourinary systems.     

Comparison of antigen expression on normal urothelial cells in tissue section and tissue culture

Antigenic characterization of urothelial cells cultured from normal adult ureter was performed. These cells were cultured using a simplified isolation and culture technique and a commercially available serum-free medium. The cells growing in these cultures had epithelioid morphology and normal quantities of DNA. The antigen expression on these cultured normal urothelial cells was evaluated using a panel of monoclonal antibodies: 5G6.4, AN43, URO-5, anti-keratin and anti-blood group antibodies, and 425 (anti-epidermal growth factor receptor). Lower levels of anti-A and AN43 binding on cultured cells were observed than are seen on urothelial cells in sections of normal ureter, while the binding of anti-blood group H, 5G6.4, and URO-5 was unchanged. Binding of anti-epidermal growth factor receptor antibody 425 was improved if the cells were grown in medium lacking epidermal growth factor. These results confirm the urothelial origin of these cultured urothelial cells but indicate that some antigenic differences between cultured normal urothelial cells and urothelial cells in situ in the normal ureter exist.     

Establishment and characterization of long-term primary mouse urothelial cell cultures

Summary Long-term mouse urothelial cell cultures were routinely established from explants of neonatal mouse bladders. Foci of proliferating cells could be observed one week after the initiation of the explant cultures. These persisted throughout the culture period and up to one year. Expression of keratin proteins confirmed the epithelial nature of the cultured cells. Morphologic analysis of nuclei sorted after DNA flow cytometry revealed a population of DNA-tetraploid and octoploid cells with large nuclei and prominent nucleoli in addition to a DNA-diploid cell population. Both cell populations showed DNA replicative activity as reflected by bromodeoxyuridine incorporation studies and mitotic activity. These long-term primary mouse urothelial cell cultures may prove useful for studies on urothelial cell kinetics and bladder carcinogenesis.     

Histomorphometry and cell kinetics of normal human bladder mucosa in vitro

Summary In this study, we have quantified the morphologic and kinetic parameters of explant cultured normal adult human urinary bladder mucosa. Quantitative parameters studied were urothelial height, cell density, labelling index and mitotic index. For these studies, urinary bladders from seven adults with no previous history of urologic disease were obtained at autopsy. Mucosal explants were maintained in rocking culture on Gelfoam rafts for up to 33 days using supplemented CMRL 1066 medium. Prior to sampling, cultures were treated with tritiated thymidine and colchicine to investigate tissue kinetics. Data was based on histologic autoradiograms. During culture, urothelial cells retained normal polarity. During the first week of culture, urothelial height increased and cell density decreased. DNA synthesis and mitotic activity occurred primarily among basal cells. DNA synthesis was first noted on day 2 of culture; mitotic activity began after 3 days of culture. Morphologically, human urothelium was well maintained; DNA synthesis and mitotic activity was variable but continued throughout culture.This work was supported by NIH Grant CA-28013     

Dysfunction of bladder urothelium and bladder urothelial cells in interstitial cystitis

The human bladder urothelium (BU) and bladder urothelial cells (BUCs) play an important role in the normal functioning of bladder including bladder storage. Current evidence in interstitial cystitis (IC) supports multiple abnormalities in bladder urothelial physiology. These data have come primarily from human studies. The discovery of a novel protein termed the antiproliferative factor (APF) uniquely expressed by IC BUCs is extremely important. APF induces increased permeability of normal BUCs grown in culture. Furthermore, APF regulates expression of other cytokines, including upregulating heparin-binding epidermal growth factor-like growth factor and downregulating epidermal growth factor by BUCs. These cytokine abnormalities were also related to increases in purinergic (adenosine triphosphate) signaling, which could mediate increased bladder sensation. Recent studies of uroplakins, which are specialized proteins expressed only in the apical urothelial cells, suggest that uroplakins play a role in the barrier function of the BU. It is also conceivable that alterations in uroplakins may result in bladder symptoms related to increased permeability or decreased protective function. As the body of knowledge about BU and BUC function increases, novel therapies targeting urothelial cells should become clinically feasible.     

Culture and characterisation of human urothelium in vivo and in vitro

The aim of this study was to culture human urothelium and generate enough cells for subsequent reconstructive surgery. Using a modification of the Rheinwald-Green method for the routine culture of keratinocytes from patients with burns, we successfully cultured 91% of 57 biopsies from the renal pelvis, ureter, bladder and urethra of paediatric patients. The cells could be split one to three up to 9 times at 7–10 day intervals, giving a surface area of 1000 cm² after a 2 month culture period. Primary cultures could not be initiated in defined medium MCDB153, although cells initiated using the Rheinwald-Green method could subsequently be propagated in this medium. Cytokeratin patterns in vitro were similar to those in vivo in the expression of keratins 7, 18 and 19 (characteristic of simple epithelia) and keratin 13 (characteristic of non-cornified stratified epithelia). Cultured urothelium also expressed keratin 14 (characteristic of cornified stratified epithelium) in about 25% of cells and keratin 16 (characteristic of fast-growing cells). These findings indicate that urothelial cells can be propagated in vitro for autologous grafting, and the next step is to identify substrates suitable for urothelial cell growth and differentiation and surgical manipulation.     

Origin of multifocal carcinomas of the bladder and upper urinary tract: Molecular analysis and clinical implications

The simultaneous or metachronous development of multifocal tumors with identical or variable histological features in the urothelial tract in a single patient is a well-known characteristic of urothelial cancer. To explain this phenomenon, two distinct concepts have been proposed: the 'field defect' hypothesis according to which urothelial cells in patients are primed to undergo transformation by previous carcinogenic insults and the 'single progenitor cell' hypothesis, which asserts that the multifocal development is caused by the seeding or intraepithelial spread of transformed cells. Results of recent molecular genetic studies support the 'single progenitor cell' hypothesis, and indicate that the genetic and phenotypic diversity observed in multifocal urothelial tumors is a consequence of clonal evolution from a single transformed cell. An understanding of the mechanism of the heterotopic recurrence of urothelial cancer may provide new prospects for early molecular detection and prevention of heterotopic recurrence of urothelial cancer.     

Bladder cell culture on small intestinal submucosa as bioscaffold: experimental study on engineered urothelial grafts

OBJECTIVES: To investigate the feasibility to perform primary urothelial cell culture using porcine small intestinal submucosa as a delivery scaffold both in vitro and after in vivo implantation in a rabbit model. MATERIALS AND METHODS: Bladder mucosa samples were aseptically obtained from a group of eight male rabbits. The mucosa was cut into fragments and placed on small intestinal submucosa matrices for selective urothelial cell culture. After complete in vitro epithelization the matrices were shaped into tubes and placed in the subcutaneous tissue and subdartos of donor rabbits. The pattern of cell growth and delivery was evaluated on retrieved grafts using histology and immunostaining at the end of the in vitro phase; then 5, 10 and 20 days after implantation. RESULTS: Histological and immunohistochemical analysis of the in vitro primary culture showed the acellular matrices covered with a thin uninterrupted monolayer of urothelial cells. The implants examined on the day 5 maintained the epithelial configuration of the cultured grafts in all samples retrieved. On the day 10 the urothelium showed increased thickness taking on a bilayer configuration. On day 20, all grafts presented the transitional cells arranged in a double layer closely resembling the natural urothelium. The immunostaining pattern displayed the maintaining of urothelial cell phenotype. No differences in epithelium growth and delivery were noted between the two sites of implantation. Five days after implantation, the histological analysis of small intestinal submucosa showed a medium degree tissue reaction with the presence of acute inflammatory cells. Angiogenesis was demonstrated by the development of several new vessels inside the matrix. After twenty days, small intestinal submucosa was gradually replaced with host tissue. CONCLUSION: The small intestinal submucosa proved to function as a means of delivering of autologous urothelial cells cultured in vitro. After ectopic in vivo implantation the bioscaffold maintained viability and growth of the surrounding cells until its degradation.     

New concepts on formation of Brunn's nests and cysts in urinary tract mucosa

To the present time, Brunn's nests have been conceived as being the result of pinching off of epithelial nests from urothelial buds. Mucosal cysts were considered to originate from Brunn's nests. In the present study, it was observed that under the influence of a strong stimulus of repair, the urothelial cells in the proximity of the focus of repair undergo numerous mitoses. The underlying connective tissue becomes loose, isolated cells of the lining urothelium “drop off” into the lamina propria in which they migrate as in a culture medium. There they multiply and form nests of cells at a distance from the lining urothelium. These migrating cells frequently have an early tendency to become arranged into cysts. Thus, during this process of repair urothelial cells behave temporarily as malignant cells.     

Short-term culture of exfoliated cells from the urine of patients with bladder tumors

Summary This report concerns the short-term culture of urothelial cells from the urine sediment of over 100 patients with bladder tumors. Primary cell outgrowth was obtained in approximately 60% of the cultures initiated. Culture outcome was not related to tumor grade, patient age, or volume of the urine sample. Around 85% of the proliferating cultures were successfully transferred into multi-compartment chamber/slides. These results suggest that the culture system may be a useful tool for the study of urothelial cells using patient material obtained by non-invasive means.